Peptides

ABSTRACT

The present invention relates to dual-site BACE1 inhibitors, their manufacture, pharmaceutical compositions containing them and their use as therapeutically active substances. The active compounds of the present invention are useful in the therapeutic and/or prophylactic treatment of e.g. Alzheimer&#39;s disease.

The present invention is concerned with peptides having dual BACE1 inhibitory properties, their manufacture, pharmaceutical compositions containing them and their use as therapeutically active substances.

TECHNICAL FIELD

The present compounds have Asp2 (β-secretase, BACE1 or Memapsin-2) inhibitory activity and may therefore be used in the therapeutic and/or prophylactic treatment of diseases and disorders characterized by elevated β-amyloid levels and/or β-amyloid oligomers and/or β-amyloid plaques and further deposits, particularly Alzheimer's disease.

BACKGROUND ART

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system and the leading cause of a progressive dementia in the elderly population. Its clinical symptoms are impairment of memory, cognition, temporal and local orientation, judgment and reasoning but also severe emotional disturbances. There are currently no treatments available which can prevent the disease or its progression or stably reverse its clinical symptoms. AD has become a major health problem in all societies with high life expectancies and also a significant economic burden for their health systems.

The BACE1 enzyme is responsible for one of the proteolytic cleavages of the APP protein that contributes to the generation of the Alzheimer's disease-associated Aβ-peptide. Retarding or stopping the production of AO-peptide through inhibition of the BACE1 enzyme is a promising therapeutic concept.

Active site-directed BACE1 inhibitors are described in e.g. WO2006/002907 and exosite-directed (catalytic domain) BACE1 inhibitors are described in e.g. Kornacker et al., Biochemistry 2005, 44, 11567-73.

Bodor et al describe modified peptides suitable to penetrate the blood-brain-barrier (Bodor et al., Science, Vol. 257, 1992).

DETAILED DESCRIPTION OF THE INVENTION

Object of the present invention is dual-site BACE1 inhibitor, binding to both, the enzymatic active site and the catalytic domain of the BACE enzyme, the preparation of the above mentioned compounds, medicaments containing them and their manufacture as well as the use of the above mentioned compounds in the therapeutic and/or prophylactic treatment of diseases and disorders which are associated with inhibition of BACE1 activity, such as Alzheimer's disease. Furthermore, the formation, or formation and deposition, of β-amyloid plaques in, on or around neurological tissue (e.g., the brain) are inhibited by the present compounds by inhibiting the Aβ production from APP or an APP fragment.

The following definitions of the general terms used in the present description apply irrespectively of whether the terms in question appear alone or in combination with other groups.

TABLE 1 amino acid abbreviations used herein Amino Acid 3-Letter 1-Letter Alanine Ala A D-Alanine DAla a Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K D-Lysine DLys k Methionine Met M Norleucine Nle — Ornithine Orn O Phenylalanine Phe F Proline Pro P D-Proline DPro p Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “Sta” stands for statine, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (CAS 49642-07-1).

The term “MetSta” stands for (3S,4S)-4-amino-3-hydroxy-6-methylthiohexanoic acid (CAS n/a), (CAS of Fmoc protected: 268542-18-3).

The term “27-OH-Chol” stands for 27-hydroxycholesterol (CAS 20380-11-4). Structure and preparation see page 18, compound 4.

The term “Chol-27-TFA-ester” stands for succinamic acid (3S,8S,9S,10R,13R,14S,17R)-17-[(1R,5R)-1,5-dimethyl-6-(2,2,2-trifluoro-acetoxy)-hexyl]-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester, this occurred as byproduct during TFA cleavage of “27-OH-Chol”-peptides.

The term “Chol‘ester”’ stands for cholesteryl hemisuccinate (CAS: 1510-21-0).

The term “PEG(3) stands for 12-amino-4,7,10-trioxadodecanoic acid (CAS: 784105-33-5).

The term “PEG(4) stands for 15-amino-4,7,10,13,tetraoxapentadecanoic acid (CAS: n/a), (CAS of Fmoc protected: 557756-85-1).

The term “pharmaceutically acceptable salts” refers to salts that are suitable for use in contact with the tissues of humans and animals. Examples of suitable salts with inorganic and organic acids are, but are not limited to acetic acid, citric acid, formic acid, fumaric acid, hydrochloric acid, lactic acid, maleic acid, malic acid, methane-sulfonic acid, nitric acid, phosphoric acid, p-toluenesulphonic acid, succinic acid, sulfuric acid, sulphuric acid, tartaric acid, trifluoroacetic acid (TFA) and the like. A specific salt is trifluoroacetate.

The terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable auxiliary substance” refer to carriers and auxiliary substances such as diluents or excipients that are compatible with the other ingredients of the formulation.

The term “pharmaceutical composition” encompasses a product comprising specified ingredients in pre-determined amounts or proportions, as well as any product that results, directly or indirectly, from combining specified ingredients in specified amounts. Preferably it encompasses a product comprising one or more active ingredients, and an optional carrier comprising inert ingredients, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.

The term “half maximal inhibitory concentration” (IC₅₀) denotes the concentration of a particular compound required for obtaining 50% inhibition of a biological process in vitro. IC₅₀ values can be converted logarithmically to pIC₅₀ values (−log IC₅₀), in which higher values indicate exponentially greater potency. The IC₅₀ value is not an absolute value but depends on experimental conditions e.g. concentrations employed. The IC₅₀ value can be converted to an absolute inhibition constant (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol. (1973) 22:3099). The term “inhibition constant” (Ki) denotes the absolute binding affinity of a particular inhibitor to a receptor. It is measured using competition binding assays and is equal to the concentration where the particular inhibitor would occupy 50% of the receptors if no competing ligand (e.g. a radioligand) was present. Ki values can be converted logarithmically to pKi values (−log Ki), in which higher values indicate exponentially greater potency.

“Therapeutically effective amount” means an amount of a compound that, when administered to a subject for treating a disease state, is sufficient to effect such treatment for the disease state. The “therapeutically effective amount” will vary depending on the compound, disease state being treated, the severity or the disease treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending medical or veterinary practitioner, and other factors.

The term “as defined herein” and “as described herein” when referring to a variable incorporates by reference the broad definition of the variable as well as preferred, more preferred and most preferred definitions, if any.

The terms “treating”, “contacting” and “reacting” when referring to a chemical reaction means adding or mixing two or more reagents under appropriate conditions to produce the indicated and/or the desired product. It should be appreciated that the reaction which produces the indicated and/or the desired product may not necessarily result directly from the combination of two reagents which were initially added, i.e., there may be one or more intermediates which are produced in the mixture which ultimately leads to the formation of the indicated and/or the desired product.

The invention also provides pharmaceutical compositions, methods of using, and methods of preparing the aforementioned compounds.

All separate embodiments may be combined.

Present invention relates to a dual-site BACE1 inhibitor, binding to both, the enzymatic active site and the catalytic domain of the BACE1 enzyme.

A certain embodiment of the invention relates to a dual-site BACE1 inhibitor according to claim 1, whereby the exosite inhibitory part (A′) is connected to the active site inhibitory part (B′) of said BACE1 inhibitor by a linker (L′), or a pharmaceutically acceptable salt thereof.

A certain embodiment of the invention relates to a dual-site BACE1 inhibitor as described herein, wherein L′ is selected from the group consisting of

-   -   i. -(Gly)_(x)-, wherein x is 3, 4, 5 or 6,     -   ii. -X-Gly-Gly-, wherein X is selected from the group consisting         of Ala, DAla, Nle, Ser, Pro, D-Pro, Lys and DLys,     -   iii. -Gly-X-Gly-, wherein X is selected from the group         consisting of Ala, DAla, Nle, Ser, Pro, D-Pro, Orn, Lys and         DLys,     -   iv. -Gly-Gly-X-, wherein X is selected from the group consisting         of Ala, DAla, Nle, Ser, Pro, D-Pro, Lys and DLys,     -   v. -Gly-DAla-DLys-,     -   vi. PEG(3), and     -   vii. PEG(4).

A certain embodiment of the invention relates to a compound as described herein, wherein A′ is selected from the group consisting of

i. Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-, ii. Tyr-Pro-Lys-Phe-Ile-Pro-Leu-,  and iii. Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-

A certain embodiment of the invention relates to a compound as described herein, wherein B′ is selected from the group consisting of

-   -   i. Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂,     -   ii. Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Y)-NH₂, wherein Y is         selected from the group consisting of Palmityl, Palmitoleic,         Oleic, Elaidic, Erucic, Vaccenic and Myristoleic, and     -   iii. Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂,

A certain embodiment of the invention relates to a compound as described herein, selected from the group consisting of

Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmitoleic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Oleic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Elaidic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Vaccenic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Erucic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Myristoleic)-NH2, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmitoleic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Oleic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Elaidic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Vaccenic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Erucic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Myristoleic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-(PEG)3-Glu-Val-Asn- MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-(PEG)4-Glu-Val-Asn- MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ala-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DAla-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DLys-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DPro-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Ala-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DAla-DLys-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DAla-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DPro-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Ala-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DAla-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DLys-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DPro-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Gly-Glu- Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Gly-Gly- Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)- NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Lys-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Nle-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Pro-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Ser-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Lys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Nle-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Orn-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Pro-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Ser-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Lys-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Nle-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Pro-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂,  and Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ser-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, or a pharmaceutical acceptable salt thereof.

A certain embodiment of the invention relates to a compound as described herein, wherein the pharmaceutically acceptable salt is trifluoroacetate.

A certain embodiment of the invention relates to a dual-site BACE1 inhibitor as described herein for use as therapeutically active substance.

A certain embodiment of the invention relates to a dual-site BACE1 inhibitor as described herein for the use as inhibitor of BACE1 activity.

A certain embodiment of the invention relates to a dual-site BACE1 inhibitor as described herein for the use as therapeutically active substance for the therapeutic and/or prophylactic treatment of diseases and disorders characterized by elevated β-amyloid levels and/or β-amyloid oligomers and/or β-amyloid plaques and further deposits, particularly Alzheimer's disease.

A certain embodiment of the invention relates to a dual-site BACE1 inhibitor as described herein for the use as therapeutically active substance for the therapeutic and/or prophylactic treatment of Alzheimer's disease.

A certain embodiment of the invention relates to a pharmaceutical composition comprising a dual-site BACE1 inhibitor as described herein and a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable auxiliary substance.

A certain embodiment of the invention relates to the use of a dual-site BACE1 inhibitor as described herein for the manufacture of a medicament for the use in inhibition of BACE1 activity.

A certain embodiment of the invention relates to the use of a dual-site BACE1 inhibitor as described herein for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of diseases and disorders characterized by elevated β-amyloid levels and/or β-amyloid oligomers and/or β-amyloid plaques and further deposits, particularly Alzheimer's disease.

A certain embodiment of the invention relates to the use of a dual-site BACE1 inhibitor as described herein for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Alzheimer's disease.

A certain embodiment of the invention relates to a dual-site BACE1 inhibitor as described herein for the use in inhibition of BACE1 activity.

A certain embodiment of the invention relates to a dual-site BACE1 inhibitor as described herein for the use in the therapeutic and/or prophylactic treatment of diseases and disorders characterized by elevated β-amyloid levels and/or β-amyloid oligomers and/or β-amyloid plaques and further deposits, particularly Alzheimer's disease.

A certain embodiment of the invention relates to a dual-site BACE1 inhibitor as described herein for the use in the therapeutic and/or prophylactic treatment of Alzheimer's disease.

A certain embodiment of the invention relates to a method for the use in inhibition of BACE1 activity, particularly for the therapeutic and/or prophylactic treatment of diseases and disorders characterized by elevated β-amyloid levels and/or β-amyloid oligomers and/or β-amyloid plaques and further deposits, Alzheimer's disease, which method comprises administering dual-site BACE1 inhibitor as described herein to a human being or animal.

Furthermore, the invention includes all optical isomers, i.e. diastereoisomers, diastereomeric mixtures, racemic mixtures, all their corresponding enantiomers and/or tautomers as well as their solvates.

The dual-site BACE1 inhibitors may be prepared as described herein. The starting material is commercially available or may be prepared in accordance with known methods.

The corresponding pharmaceutically acceptable salts with acids can be obtained by standard methods known to the person skilled in the art, e.g. by dissolving the dual-site BACE1 inhibitor in a suitable solvent such as e.g. dioxan or THF and adding an appropriate amount of the corresponding acid. The products can usually be isolated by filtration or by chromatography. The conversion of a dual-site BACE1 inhibitor into a pharmaceutically acceptable salt with a base can be carried out by treatment of such a compound with such a base. One possible method to form such a salt is e.g. by addition of 1/n equivalents of a basic salt such as e.g. M(OH)_(n), wherein M=metal or ammonium cation and n=number of hydroxide anions, to a solution of the compound in a suitable solvent (e.g. ethanol, ethanol-water mixture, tetrahydrofuran-water mixture) and to remove the solvent by evaporation or lyophilisation.

Insofar as their preparation is not described in the examples, the dual-site BACE1 inhibitors as well as all intermediate products can be prepared according to analogous methods or according to the methods set forth herewithin. Starting materials are commercially available, known in the art or can be prepared by methods known in the art or in analogy thereto.

It will be appreciated that the dual-site BACE1 inhibitors in this invention may be derivatised at functional groups to provide derivatives which are capable of conversion back to the parent compound in vivo.

Pharmacological Tests

The dual-site BACE1 inhibitor and their pharmaceutically acceptable salts possess valuable pharmacological properties. It has been found that the compounds of the present invention are associated with inhibition of BACE1 activity. The compounds were investigated in accordance with the test given hereinafter.

Cellular Aβ-Lowering Assay:

Human HEK293 cells which are stably transfected with a vector expressing a cDNA of the human APP wt gene (APP695) were used to assess the potency of the compounds in a cellular assay. The cells were seeded in 96-well microtiter plates in cell culture medium (Iscove, plus 10% (v/v) fetal bovine serum, glutamine, penicillin/streptomycin) to about 80% confluence and the compounds were added at a 10× concentration in 1/10 volume of medium without FCS containing 8% DMSO (final concentration of DMSO was kept at 0.8% v/v). After 18-20 hrs incubation at 37° C. and 5% CO₂ in a humidified incubator the culture supernatant was harvested for the determination of Aβ40 concentrations. 96well ELISA plates (e.g., Nunc MaxiSorb) were coated with monoclonal antibody which specifically recognize the C-terminal end of Aβ40 (Brockhaus et al., NeuroReport 9, 1481-1486; 1998). After blocking of non-specific binding sites with e.g. 1% BSA and washing, the culture supernatants were added in suitable dilutions together with a horseradish peroxidase-coupled Aβ detection antibody (e.g., antibody 4G8, Senetek, Maryland Heights, Mo.) and incubated for 5 to 7 hrs. Subsequently the wells of the microtiter plate were washed extensively with Tris-buffered saline containing 0.05% Tween 20 and the assay was developed with tetramethylbenzidine/H₂O₂ in citric acid buffer. After stopping the reaction with one volume 1 N H₂SO₄ the reaction was measured in an ELISA reader at 450 nm wavelength. The concentrations of Aβ in the culture supernatants were calculated from a standard curve obtained with known amounts of pure Aβ peptide.

TABLE 2 IC₅₀ values of selected examples IC 50 Ex. Name Systematic Name MW (μM)  1 YPYFIPL-GGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2508.9 0.12 Sta-VAEFK(Palm)- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA  2 YPYFIPL-AGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ala- 2523.9 0.053 Sta-VAEFK(Palm)- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA  3 YPYFIPL-GAG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2522.9 0.095 Sta-VAEFK(Palm)- Ala-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA  4 YPYFIPL-GGA-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2522.9 0.084 Sta-VAEFK(Palm)- Gly-Ala-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA  5 YPYFIPL-Nle-GG- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Nle- 2565.0 0.081 EVN-Sta- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- VAEFK(Palm)-NH₂ x Glu-Phe-Lys(Palmityl)-NH₂ x TFA TFA  6 YPYFIPL-G-N1e-G- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2565.0 0.12 EVN-Sta- Nle-Gly-Glu-Val-Asn-Sta-Val-Ala- VAEFK(Palm)-NH₂ x Glu-Phe-Lys(Palmityl)-NH₂ x TFA TFA  7 YPYFIPL-GG-Nle- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2565.0 0.18 EVN-Sta- Gly-Nle-Glu-Val-Asn-Sta-Val-Ala- VAEFK(Palm)-NH₂ x Glu-Phe-Lys(Palmityl)-NH₂ x TFA TFA  8 YPYFIPL-SGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ser- 2538.9 0.036 Sta-VAEFK(Palm)- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA  9 YPYFIPL-GSG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2538.9 0.041 Sta-VAEFK(Palm)- Ser-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA 10 YPYFIPL-GGS-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2538.9 0.053 Sta-VAEFK(Palm)- Gly-Ser-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA 11 YPYFIPL-aGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DAla- 2522.9 0.013 Sta-VAEFK(Palm)- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA 12 YPYFIPL-GaG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2522.9 0.0086 Sta-VAEFK(Palm)- DAla-Gly-Glu-Val-Asn-Sta-Val- NH₂ x TFA Ala-Glu-Phe-Lys(Palmityl)-NH₂ x TFA 13 YPYFIPL-GGa-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2522.9 0.025 Sta-VAEFK(Palm)- Gly-DAla-Glu-Val-Asn-Sta-Val- NH₂ x TFA Ala-Glu-Phe-Lys(Palmityl)-NH2 x TFA 14 YPYFIPL-PGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Pro- 2549.0 0.021 Sta-VAEFK(Palm)- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA 15 YPYFIPL-GPG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2549.0 0.14 Sta-VAEFK(Palm)- Pro-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA 16 YPYFIPL-GGP-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2549.0 0.049 Sta-VAEFK(Palm)- Gly-Pro-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA 17 YPYFIPL-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2580.0 0.064 Sta-VAEFK(Palm)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x TFA Ala-Glu-Phe-Lys(Palmityl)-NH₂ x TFA 18 YPYFIPL-pGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DPro- 2549.0 0.050 Sta-VAEFK(Palm)- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA 19 YPYFIPL-GpG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2549.0 0.048 Sta-VAEFK(Palm)- DPro-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA 20 YPYFIPL-GGp-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2549.0 0.11 Sta-VAEFK(Palm)- Gly-DPro-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFA Glu-Phe-Lys(Palmityl)-NH₂ x TFA 21 YPYFIPL-Gak-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2594.1 0.098 Sta-VAEFK(Palm)- DAla-DLys-Glu-Val-Asn-Sta-Val- NH₂ x TFA Ala-Glu-Phe-Lys(Palmityl)-NH₂ x TFA 22 YPYFIPL-GGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2458.9 0.060 Sta-VAEpK(Palm)-NH₂ Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-  x TFA Glu-DPro-Lys(Palmityl)-NH₂ x TFA 23 YPYFIPL-GKG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2644.0 0.078 Sta-VAE-DPro- Lys-Gly-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH2 x 2TFA Glu-DPro-Lys(Palm)-NH₂ x 2TFA 24 YPYFIPL-GOG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2630.0 0.067 Sta-VAE-DPro- Orn-Gly-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH2 x 2TFA Glu-DPro-Lys(Palm)-NH₂ x 2TFA 25 YPYFIPL-KGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Lys- 2644.0 0.0039 Sta-VAE-DPro- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH₂ x 2TFA Glu-DPro-Lys(Palm)-NH₂ x 2TFA 26 YPYFIPL-GGK-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2644.0 0.0033 Sta-VAE-DPro- Gly-Lys-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH₂ x 2TFA Glu-DPro-Lys(Palm)-NH₂ x 2TFA 27 YPYFIPL-kGG-EVN- Tyr-Pro-Tyr-Phe-fle-Pro-Leu-DLys- 2644.0 0.0060 Sta-VAE-DPro- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH₂ x 2TFA Glu-DPro-Lys(Palm)-NH₂ x 2TFA 28 YPYFIPL-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2644.0 0.0029 Sta-VAE-DPro- DLys-Gly-Glu-Val-Asn-Sta-Val- K(Palm)-NH₂ x 2TFA Ala-Glu-DPro-Lys(Palm)-NH₂ x 2TFA 29 YPYFIPL-GGk-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2644.0 0.0057 Sta-VAE-DPro- Gly-DLys-Glu-Val-Asn-Sta-Val- K(Palm)-NH₂ x 2TFA Ala-Glu-DPro-Lys(Palm).NH₂ x 2TFA 30 YPYFIPL-GGGG- Tyr-Pro-Tyr-Phe-fle-Pro-Leu-Gly- 2584.0 0.33 EVN-MetSta-VAE- Gly-Gly-Gly-Glu-Val-Asn-MetSta- DPhe-K(Palm)-NH₂ x Val-Ala-Glu-DPhe-Lys(Palm)-NH₂ x TFA TFA 31 YPYFIPL-GGGGG- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2641.1 0.034 EVN-MetSta-VAE- Gly-Gly-Gly-Gly-Glu-Val-Asn- DPhe-K(Palm)-NH₂ x MetSta-Val-Ala-Glu-DPhe- TFA Lys(Palm)-NH₂ x TFA 32 YPYFIPL-PEG(4)- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu- 2603.1 0.018 EVN-MetSta-VAE- (PEG)4-Glu-Val-Asn-MetSta-Val- DPhe-K(Palm)-NH₂ x Ala-Glu-DPhe-Lys(Palm)-NH₂ x TFA TFA 33 YPYFIPL-PEG(3)- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu- 2559.0 0.041 EVN-MetSta-VAE- (PEG)3-Glu-Val-Asn-MetSta-Val- DPhe-K(Palm)-NH₂ x Ala-Glu-DPhe-Lys(Palm)-NH₂ x TFA TFA 34 YPKFIPL-GkG-EVN- Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2723.0 0.00137 Sta-VAEp-K(Palm)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Palm)-NH₂ x 3TFA 35 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2773.0 0.000199 Sta-VAEp-K(Palm)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Palm)-NH₂ x 3TFA 36 YPKFIPL-GkG-EVN- Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2721.0 0.0199 Sta-VAEp-K DLys-Gly-Glu-Val-Asn-Sta-Val- (Palmitoleic)-NH₂ x Ala-Glu-DPro-Lys(Palmitoleic)-NH₂ 3TFA x 3TFA 37 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2771.0 0.00428 Sta-VAEp- DLys-Gly-Glu-Val-Asn-Sta-Val- K(Palmitoleic)-NH₂ x Ala-Glu-DPro-Lys(Palmitoleic)-NH₂ 3TFA x 3TFA 38 YPKFIPL-GkG-EVN- Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2749.1 0.00602 Sta-VAEp-K(Oleic)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Oleic)-NH₂ x 3TFA 39 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2799.1 0.00142 Sta-VAEp-K(Oleic)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Oleic)-NH₂ x 3TFA 40 YPKFIPL-GkG-EVN- Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2749.1 0.00302 Sta-VAEp-K(Elaidic)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Elaidic)-NH₂ x 3TFA 41 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2799.1 0.000906 Sta-VAEp-K(Elaidic)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Elaidic)-NH₂ x 3TFA 42 YPKFIPL-GkG-EVN- Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2749.1 0.00268 Sta-VAEp-K DLys-Gly-Glu-Val-Asn-Sta-Val- (Vaccenic)-NH₂ x Ala-Glu-DPro-Lys(Vaccenic)-NH₂ x 3TFA 3TFA 43 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2799.1 0.00111 Sta-VAEp- DLys-Gly-Glu-Val-Asn-Sta-Val- K(Vaccenic)-NH₂ x Ala-Glu-DPro-Lys(Vaccenic)-NH₂ x 3TFA 3TFA 44 YPKFIPL-GkG-EVN- Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2805.2 0.00115 Sta-VAEp-K(Erucic)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Erucic)-NH₂ x 3TFA 45 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2855.2 0.000147 Sta-VAEp-K(Erucic)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Erucic)-NH₂ x 3TFA 46 YPKFIPL-GkG-EVN- Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2693.0 0.208 Sta-VAEp-K DLys-Gly-Glu-Val-Asn-Sta-Val- (Myristoleic)-NH₂ x Ala-Glu-DPro-Lys(Myristoleic)-NH₂ 3TFA  x 3TFA 47 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2743.0 0.0196 Sta-VAEp-K DLys-Gly-Glu-Val-Asn-Sta-Val- (Myristoleic)-NH₂ x Ala-Glu-DPro-Lys(Myristoleic)-NH₂ 3TFA  x 3TFA

Pharmaceutical Compositions

The dual-site BACE1 inhibitors and the pharmaceutically acceptable salts can be used as therapeutically active substances, e.g. in the form of pharmaceutical preparations. Examples for pharmaceutical preparations are an enteral formulation, e.g. in the form of tablets, coated tablets, dragées, hard and soft gelatine capsules, solutions, emulsions or suspensions or the like. The administration can, however, also be effected rectally, e.g. in the form of suppositories, parenterally, e.g. in the form of injection solutions, or as intranasal delivery, e.g. as nasal spray.

The dual-site BACE1 inhibitors and the pharmaceutically acceptable salts thereof can be processed with pharmaceutically inert, inorganic or organic carriers for the production of pharmaceutical preparations. Lactose, corn starch or derivatives thereof, talc, stearic acids or its salts and the like can be used, for example, as such carriers for tablets, coated tablets, dragées and hard gelatine capsules. Suitable carriers for soft gelatine capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols and the like. Depending on the nature of the active substance no carriers are however usually required in the case of soft gelatine capsules. Suitable carriers for the production of solutions and syrups are, for example, water, polyols, glycerol, vegetable oil and the like. Suitable carriers for suppositories are, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like. The dual-site BACE1 inhibitors and the pharmaceutically acceptable salts thereof can also be encapsulated in suitable polymers or formulated using nanotechnology.

The pharmaceutical preparations can, moreover, contain pharmaceutically acceptable auxiliary substances such as preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.

Medicaments containing a dual-site BACE1 inhibitor or a pharmaceutically acceptable salt thereof and a therapeutically inert carrier are also an object of the present invention, as is a process for their production, which comprises bringing one or more dual-site BACE1 inhibitors and/or pharmaceutically acceptable salts thereof and, if desired, one or more other therapeutically valuable substances into a galenical administration form together with one or more therapeutically inert carriers.

The dosage can vary within wide limits and will, of course, have to be adjusted to the individual requirements in each particular case. In the case of oral administration the dosage for adults can vary from about 0.01 mg to about 1000 mg per day of a dual-site BACE1 inhibitors or of the corresponding amount of a pharmaceutically acceptable salt thereof. The daily dosage may be administered as single dose or in divided doses and, in addition, the upper limit can also be exceeded when this is found to be indicated.

The following examples illustrate the present invention without limiting it, but serve merely as representative thereof. The pharmaceutical preparations conveniently contain about 1-500 mg, preferably 1-100 mg, of a dual-site BACE1 inhibitor. Examples of compositions according to the invention are:

Example A

Tablets of the following composition are manufactured in the usual manner:

TABLE 3 possible tablet composition mg/tablet ingredient 5 25 100 500 Dual-site BACE1 inhibitor 5 25 100 500 Lactose Anhydrous DTG 125 105 30 150 Sta-Rx 1500 6 6 6 60 Microcrystalline Cellulose 30 30 30 450 Magnesium Stearate 1 1 1 1 Total 167 167 167 831

Manufacturing Procedure

1. Mix ingredients 1, 2, 3 and 4 and granulate with purified water. 2. Dry the granules at 50° C. 3. Pass the granules through suitable milling equipment. 4. Add ingredient 5 and mix for three minutes; compress on a suitable press.

Example B-1

Capsules of the following composition are manufactured:

TABLE 4 possible capsule ingredient composition mg/capsule ingredient 5 25 100 500 Dual-site BACE1 inhibitor 5 25 100 500 Hydrous Lactose 159 123 148 — Corn Starch 25 35 40 70 Talk 10 15 10 25 Magnesium Stearate 1 2 2 5 Total 200 200 300 600

Manufacturing Procedure

1. Mix ingredients 1, 2 and 3 in a suitable mixer for 30 minutes. 2. Add ingredients 4 and 5 and mix for 3 minutes. 3. Fill into a suitable capsule.

The dual-site BACE1 inhibitor, lactose and corn starch are firstly mixed in a mixer and then in a comminuting machine. The mixture is returned to the mixer; the talc is added thereto and mixed thoroughly. The mixture is filled by machine into suitable capsules, e.g. hard gelatine capsules.

Example B-2

Soft Gelatine Capsules of the following composition are manufactured:

TABLE 5 possible soft gelatin capsule ingredient composition ingredient mg/capsule Dual-site BACE1 inhibitor 5 Yellow wax 8 Hydrogenated Soya bean oil 8 Partially hydrogenated plant oils 34 Soya bean oil 110 Total 165

TABLE 6 possible soft gelatin capsule composition ingredient mg/capsule Gelatin 75 Glycerol 85% 32 Karion 83 8 (dry matter) Titan dioxide 0.4 Iron oxide yellow 1.1 Total 116.5

Manufacturing Procedure

The dual-site BACE1 inhibitor is dissolved in a warm melting of the other ingredients and the mixture is filled into soft gelatin capsules of appropriate size. The filled soft gelatin capsules are treated according to the usual procedures.

Example C

Suppositories of the following composition are manufactured:

TABLE 7 possible suppository composition ingredient mg/supp. Dual-site BACE1 inhibitor 15 Suppository mass 1285 Total 1300

Manufacturing Procedure

The suppository mass is melted in a glass or steel vessel, mixed thoroughly and cooled to 45° C. Thereupon, the finely powdered dual-site BACE1 inhibitor is added thereto and stirred until it has dispersed completely. The mixture is poured into suppository moulds of suitable size, left to cool; the suppositories are then removed from the moulds and packed individually in wax paper or metal foil.

Example D

Injection solutions of the following composition are manufactured:

TABLE 8 possible injection solution composition ingredient mg/injection solution. Dual-site BACE1 inhibitor  3 Polyethylene Glycol 400 150 acetic acid q.s. ad pH 5.0 water for injection solutions ad 1.0 ml

Manufacturing Procedure

The dual-site BACE1 inhibitor is dissolved in a mixture of Polyethylene Glycol 400 and water for injection (part). The pH is adjusted to 5.0 by acetic acid. The volume is adjusted to 1.0 ml by addition of the residual amount of water. The solution is filtered, filled into vials using an appropriate overage and sterilized.

Example E

Sachets of the following composition are manufactured:

TABLE 9 possible sachet composition ingredient mg/sachet Dual-site BACE1 inhibitor 50 Lactose, fine powder 1015 Microcrystalline cellulose (AVICEL PH 102) 1400 Sodium carboxymethyl cellulose 14 Polyvinylpyrrolidon K 30 10 Magnesium stearate 10 Flavoring additives 1 Total 2500

Manufacturing Procedure

The dual-site BACE1 inhibitor is mixed with lactose, microcrystalline cellulose and sodium carboxymethyl cellulose and granulated with a mixture of polyvinylpyrrolidone in water. The granulate is mixed with magnesium stearate and the flavoring additives and filled into sachets.

Experimental Part

The following examples are provided for illustration of the invention. They should not be considered as limiting the scope of the invention, but merely as being representative thereof.

General Procedures for the CEM Liberty Microwave Peptide Synthesizer:

0.1 mMol scale:

Deprotection of Fmoc:

The washed and preswelled resin (435 mg, 0.1 mMol, TentaGel S RAM (Load: 0.23 mMol/g), (Rapp Polymere, Cat: S30023) was treated with a solution of piperidine 20% in dimethylformamide (DMF) (7.0 mL) under microwave condition at 50° C. for 3 minutes for initial deprotection. The resin was washed with DMF and treated with a solution of piperidine 20% in DMF (7.0 mL) under microwave condition at 75° C. for 5 minutes for deprotection.

Coupling of amino acids:

To the washed and preswelled resin was added a solution of amino acid, 0.2M in DMF (2.5 mL, 5.0 eq.) followed by a solution of COMU® 0.5M in DMF (1.0 mL, 5.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) followed by a solution of diisopropylethylamine (DIPEA) 2M in N-Methyl-2-pyrrolidone (NMP) (0.5 mL, 10.0 eq.). This reaction mixture was treated under microwave condition at 75° C. for 5 minutes for coupling.

0.25 mMol scale:

Deprotection of Fmoc:

The washed and preswelled resin (1.09 g, 0.25 mMol, TentaGel S RAM (Load: 0.23 mMol/g), (Rapp Polymere, Cat: S30023) was treated with a solution of piperidine 20% in DMF (10.0 mL) under microwave condition at 50° C. for 3 minutes for initial deprotection. The resin was washed with DMF and treated with a solution of piperidine 20% in DMF (10.0 mL) under microwave condition at 75° C. for 5 minutes for deprotection.

Coupling of amino acids:

To the washed and preswelled resin was added a solution of amino acid, 0.2M in DMF (5.0 mL, 4.0 eq.) followed by a solution of COMU® 0.5M in DMF (2.0 mL, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) followed by a solution of DIPEA 2M in NMP (1.0 mL, 8.0 eq.). This reaction mixture was treated under microwave condition at 75° C. for 5 minutes for coupling.

General Procedure for Coupling of “n-C8”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with a solution of caprylic acid (79.2 μl, 5.0 eq.), (CAS: 124-07-2, Fluka) and COMU® (214 mg, 5.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) and DIPEA (204 μL, 12.0 eq.) in 5.0 mL DMF for 1 hour at room temperature on the shaker.

General Procedure for Coupling of “n-C12”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with a solution of dodecanoic acid (100 mg, 5.0 eq.), (CAS: 143-07-7, Aldrich) and COMU® (214 mg, 5.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) and DIPEA (204 μL, 12.0 eq.) in 5.0 mL DMF for 1 hour at room temperature on the shaker.

General Procedure for Coupling of “n-C14”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with a solution of myristic acid (114 mg, 5.0 eq.), (CAS: 544-63-8, Fluka) and COMU® (214 mg, 5.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) and DIPEA (204 μL, 12.0 eq.) in 5.0 mL DMF for 1 hour at room temperature on the shaker.

General Procedure for Coupling of “Palmitoleic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with a solution of palmitoleic acid (102 mg, 4.0 eq.), (CAS: 373-49-9) and COMU® (176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperature on the shaker.

General Procedure for Coupling of “Oleic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with a solution of oleic acid (113 mg, 4.0 eq.), (CAS: 112-80-1) and COMU® (176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperature on the shaker.

General Procedure for Coupling of “Elaidic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with a solution of elaidic acid (113 mg, 4.0 eq.), (CAS: 112-79-8) and COMU® (176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperature on the shaker.

General Procedure for Coupling of “Vaccenic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with a solution of vaccenic acid (113 mg, 4.0 eq.), (CAS: 693-72-1) and COMU® (176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperature on the shaker.

General Procedure for Coupling of “Erucic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with a solution of erucic acid (113 mg, 4.0 eq.), (CAS: 112-86-7) and COMU® (176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperature on the shaker.

General Procedure for Coupling of “Myristoleic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with a solution of myristoleic acid (90.5 mg, 4.0 eq.), (CAS: 544-64-9) and COMU® (176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperature on the shaker.

General Procedure for Final Cleavage:

0.1 mMol scale:

The resin was washed with dichloromethane (CH₂Cl₂) and then treated with a solution of trifluoroacetic acid (TFA): TIS:water 95:2.5:2.5 (5 mL) for 30 minutes at room temperature on the shaker. The resin was filtered. The crude peptide was precipitated with diethylether (Et₂O) (35 mL). The suspension was centrifuged and the solvent was decanted. The solid was dissolved in acetonitrile and water and freeze-dried to get the crude peptide.

General Procedure for Purification:

The crude product was dissolved in acetonitrile and water (containing 0.1% TFA) and then purified by preparative HPLC. Column YMC-Actus Pro C8, 5 μm, 75×30 mm with a gradient of water (containing 0.1% TFA):acetonitrile 70:30 to 2:98 and with a flow of 30 mL/min.

All Examples as described in table 1 can be prepared analogous to the general procedures described herein. 

1. A dual-site BACE1 inhibitor, binding to both, the enzymatic active site and the catalytic domain of the BACE enzyme.
 2. The dual-site BACE1 inhibitor according to claim 1, whereby the exocite inhibitory part (A′) is connected to the active site inhibitory part (B′) of said BACE1 inhibitor by a linker (L′), or a pharmaceutically acceptable salt thereof.
 3. The dual-site BACE1 inhibitor according to claim 2, wherein L′ is selected from the group consisting of: i. -(Gly)_(x)-, wherein x is 3, 4, 5 or 6, ii. -X-Gly-Gly-, wherein X is selected from the group consisting of Ala, DAla, Nle, Ser, Pro, D-Pro, Lys and DLys, iii. -Gly-X-Gly-, wherein X is selected from the group consisting of Ala, DAla, Nle, Ser, Pro, D-Pro, Orn, Lys and DLys, iv. -Gly-Gly-X-, wherein X is selected from the group consisting of Ala, DAla, Nle, Ser, Pro, D-Pro, Lys and DLys, v. -Gly-DAla-DLys-, vi. PEG(3), and vii. PEG(4).
 4. The dual-site BACE1 inhibitor according to claim 2, wherein A′ is selected from the group consisting of:
 1. Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-, ii Tyr-Pro-Lys-Phe-Ile-Pro-Leu-, and iii. Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-.


5. The dual-site BACE1 inhibitor according to claim 2, wherein B′ is selected from the group consisting of i. Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, ii. Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Y)-NH₂, wherein Y is selected from the group consisting of Palmityl, Palmitoleic, Oleic, Elaidic, Erucic, Vaccenic and Myristoleic, and iii. Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Pam)-NH₂.
 6. The dual-site BACE1 inhibitor according to claim 1, selected from the group consisting of: Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmitoleic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Oleic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Elaidic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Vaccenic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Erucic)-NH₂, Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Myristoleic)-NH2, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmitoleic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Oleic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Elaidic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Vaccenic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Erucic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Myristoleic)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-(PEG)3-Glu-Val-Asn- MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-(PEG)4-Glu-Val-Asn- MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ala-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DAla-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DLys-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DPro-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Ala-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DAla-DLys-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DAla-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DPro-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Ala-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DAla-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DLys-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DPro-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Gly-Glu- Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Gly-Gly- Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)- NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Lys-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Nle-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Pro-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Ser-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Lys-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Nle-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Orn-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Pro-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Ser-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Lys-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Nle-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Pro-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂,  and Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ser-Gly-Gly-Glu-Val- Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂,


7. The dual-site BACE1 inhibitor according to claim 6, wherein the pharmaceutically acceptable salt is trifluoroacetate.
 8. (canceled)
 9. (canceled)
 10. A pharmaceutical composition, comprising a dual-site BACE1 inhibitor according to claim 1 and a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable auxiliary substance.
 11. (canceled)
 12. A method of inhibiting BACE1 activity, comprising the step of administering a dual-site BACE1 inhibitor according to claim 1 to a human being or animal in need thereof.
 13. (canceled)
 14. A method of treating a disease or disorder characterized by elevated β-amyloid levels and/or β-amyloid oligomers and/or β-amyloid plaques and further deposits, comprising the step of administering a dual-site BACE1 inhibitor according to claim 1 to a human being or animal in need thereof.
 15. The method according to claim 14, wherein said disease or disorder is Alzheimer's disease, diabetes or type 2 diabetes.
 16. A method of treating Alzheimer's disease, comprising the step of administering a dual-site BACE1 inhibitor according to claim 1 to a human being or animal in need thereof.
 17. A method of treating diabetes, comprising the step of administering a dual-site BACE1 inhibitor according to claim 1 to a human being or animal in need thereof.
 18. The method according to claim 17, wherein said diabetes is type 2 diabetes. 